The proliferation-associated nuclear protein Ki-67 in the bovine system: partial characterisation and its application for determination of the proliferation of Theileria -infected bovine cells

Abstract

Theileria annulata-infected bovine cells as well as mitogen-stimulated bovine peripheral blood mononuclear cells (PBMC) express a proliferation-associated nuclear protein equivalent to the human Ki-67 protein. In analogy to the human system, the expression of the bovine Ki-67 protein is restricted to proliferating cells only, since (a) Ki-67 expression paralleled [3H]-thymidine incorporation in concanavalin A (Con A)-stimulated bovine PBMC, (b) Ki-67 was not detectable in quiescent bovine cells, and (c) Ki-67 expression in Theileria-infected cells is related to the presence of the parasites within the cytoplasm of the host cells; upon treatment with the theilericidal drug buparvaquone the parasites are destroyed and the cells cease to proliferate and to express the Ki-67 protein. Western-blot analysis of lysates of proliferating bovine cells revealed that the prototype monoclonal antibody Ki-67 and the new equivalent antibody MIB-1 detected one prominent protein band with an apparent molecular weight of 430 kDa. Two cDNA clones (pUC18.B1.Ki-67 and pUC18.B2.Ki-67) were isolated from a lambdagt11 cDNA library of T. annulata-infected bovine cells by immunoscreening with the monoclonal antibody MIB-1. Comparison of these cDNA sequences with those of the human Ki-67 protein revealed 60-70% identity. Within the "Ki-67 motif", identity proved to be 80% at the amino acid level. The remarkable identity between bovine and human Ki-67 proteins suggests that MIB-1 can be used as a marker for cell proliferation in animal research. In this context we could identify proliferating cells in lymph nodes of Theileria-infected animals and, furthermore, we could distinguish between infected and uninfected proliferating cells using MIB-1 and an antiserum against a recombinant parasite protein designated SA288.

Cytoplasmic sequestration of p53 promotes survival in leukocytes transformed by Theileria

“…For buparvaquone treatment, which specifically interacts with the mitochondrial electron transport chain of the parasite (McHardy and Morgan, 1985) and does not influence the vitality of ConA-stimulated bovine PBMCs (Shayan et al, 1999), we incubated 1 Â 10 7 cells in 25 ml medium and 0.05 mg/ ml buparvaquone up to the desired time points.…”

“…The schizont stage of the intracellular apicomplexan parasite Theileria annulata, which causes tropical theileriosis in cattle, shares the unique property of leukoproliferative Theileria to immortalize its leukocyte host cells such that they undergo clonal expansion and acquire characteristics of metastatic tumor cells (Ahmed et al, 1999). Importantly, Theileria-induced transformation is reversible and parasite dependent, because specific elimination of the parasites from infected cells by the naphthoquinone derivate buparvaquone results in cessation of proliferation and apoptosis of the host cell (McHardy and Wekesa, 1985;Dobbelaere et al, 1988;Shayan et al, 1999). It is thus postulated that the parasite interferes with mitogenic pathways of the host cell, and several pathways, including the stress-dependent c-Jun kinase pathway (Galley et al, 1997), the mitogen-activated protein kinase pathway and the phosphoinositide-3 kinase/ protein kinase-B pathway, have been investigated (Heussler et al, 2001;Seitzer et al, 2006).…”

Simultaneous differentiation between Theileria spp. and Babesia spp. on stained blood smear using PCR

“…In certain cases, serological methods such as the immune fluorescence antibody test (IFAT) or immunoperoxidase test have also been applied (Jianxus and Hung 1997;Leemans et al 1997;Shayan et al 1999).…”

Theileria annulatasurface protein (TaSP) is a target of cyclin‐dependent kinase 1 phosphorylation in Theileria annulata‐infected cells

“…The schizont interacts with the mitotic machinery of the host cell to ensure its own propagation to new daughter cells. Since CDK1 is a key molecule during mitosis, we addressed the impact of CDK1 inhibition on Theileria-infected cells: adhered TaA288 cells were treated for 24 hr with 10 or 20 µM purvalanol A (purvA), a highly specific inhibitor for CDK1 (and CDK2) (Buquet-Fagot, Lallemand, Montagne, & Mester, 1997;Gray, Détivaud, Doerig, & Meijer, 1999;Gray et al, 1998;Villerbu, Gaben, Redeuilh, & Mester, 2002) ( Figure 6a independently confirm that reduced BrdU signals correspond to impaired proliferation, we used the proliferation marker Ki-67 (Shayan et al, 1999) to determine the percentage of proliferating cells after purvA treatment (Figure 6c). Therefore, TaA288 cells were grown on cover slips and treated with 10 µM purvA.…”